Senescent endothelial cells are predisposed to SARS-CoV-2 infection and subsequent endothelial dysfunction

The coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains to spread worldwide. COVID-19 is characterized by the striking high mortality in elderly; however, its mechanistic insights remain unclear. Systemic thrombosis has been highlighted in the pathogenesis of COVID-19, and lung microangiopathy in association with endothelial cells (ECs) injury has been reported by post-mortem analysis of the lungs. Here, we experimentally investigated the SARS-CoV-2 infection in cultured human ECs, and performed a comparative analysis for post-infection molecular events using early passage and replicative senescent ECs. We found that; (1) SARS-CoV-2 infects ECs but does not replicate and disappears in 72 hours without causing severe cell damage, (2) Senescent ECs are highly susceptible to SARS-CoV-2 infection, (3) SARS-CoV-2 infection alters various genes expression, which could cause EC dysfunctions, (4) More genes expression is affected in senescent ECs by SARS-CoV-2 infection than in early passage ECs, which might causes further exacerbated dysfunction in senescent ECs. These data suggest that sustained EC dysfunctions due to SARS-CoV-2 infection may contribute to the microangiopathy in the lungs, leading to deteriorated inflammation and thrombosis in COVID-19. Our data also suggest a possible causative role of EC senescence in the aggravated disease in elder COVID-19 patients.

replicative senescent (P20) HUVECs (n = 4 each). Target genes expression was normalized to 18S expression levels. (B) Quantitative PCR analysis for ACE2 in Calu-3 cells and early passage or replicative senescent ECs (n = 3 each). ACE2 expression was normalized to 18S expression levels. (C) Quantitative PCR analysis for SARS-CoV-2 ORF1ab in early passage and replicative senescent ECs infected with SARS-CoV-2 at 1 MOI (n = 4 for senescent EC-no infection; n = 6 each for others). ORF1ab expression was normalized to 18S expression levels.
(D) Representative images of immunocytochemistry for SARS-CoV-2 spike protein (red fluorescence) in early passage and replicative senescent HUVECs infected with SARS-CoV-2 (50 MOI) at 6 hpi. Bars: 100 μm. Spikestaining was quantitatively analyzed (n = 12 each). (E) SARS-CoV-2 attachment on early passage and replicative senescent HUVECs was analyzed using quantitative PCR for ORF1ab (n = 6 each). Statistical analyses were performed using two-tailed unpaired Student's t-test (A,B), while Mann Whitney U-test were used for D and E. One-way ANOVA with Fisher's LSD post hoc test was used for statistical analysis between multiple groups (C). Data are presented as mean ± SE *P < 0.05, ****P < 0.0001 and #Not significant.  10 .
To investigate the SARS-CoV-2 infection in ECs, we incubated these HUVECs with SARS-CoV-2 at 1 MOI for 1 h, followed by incubation with fresh growth medium. RNAs were collected from the cells, and the SARS-CoV-2 infection was assessed by analyzing virus RNA (open reading frame1 ab; ORF1ab) levels using quantitative RT-PCR 23,24 . SARS-CoV-2 RNA was detected in cellular RNAs, indicating the virus entry into ECs (Fig. 1C). Of note, SARS-CoV-2 RNA levels were significantly higher in senescent HUVECs than in early passage cells, especially at early time point after infection (Fig. 1C). However, SARS-CoV-2 RNA levels in the cells substantially reduced and became indetectable at 72 h post infection (hpi) (Fig. 1C). We further assessed virus infection by immunostaining for SARS-CoV-2 spike protein. Consistent with the virus RNA qPCR analysis, significant SARS-CoV-2 spike staining was observed in senescent HUVECs, while the spike staining was barely detectable in early passage HUVECs (Fig. 1D).
Because of the remarkable difference of virus RNA levels at early time of infection, we performed a virus attachment assay. SARS-CoV-2 attachment was not different between early passage and senescent HUVECs (Fig. 1E). Therefore, virus entry after attachment should be enhanced in replicative senescent HUVECs comparing to that in early passage cells. These data collectively suggest that SARS-CoV-2 indeed infects ECs, and EC cellular senescence enhances virus infection, while SARS-CoV-2 seems not to replicate in ECs and disappears from the cells soon.

SARS-CoV-2 enters into ECs through endocytosis.
We further confirmed SARS-CoV-2 infection by detecting intracellular virions using transmission electron microscopy. Intracellular virions surrounded by endosome-like structure were detected in senescent HUVECs infected with SARS-CoV-2 at 2 hpi ( Fig. 2A). At 6 hpi, many SARS-CoV-2 entry via caveolae-like structures was observed in senescent HUVECs, while much less virus entry was detected in early passage HUVECs (Fig. 2B). These observations are consistent with the remarkable increase of virus RNA in senescent HUVECs at early time point after infection. Observation at later time point revealed that virions accumulated in endosome-like structures at 24 and 48 hpi, and they appeared to be disrupted at 48 hpi (Fig. 2C). We then performed double staining for SARS-CoV-2 spike protein and Rab5A, an endosome marker protein, and found that virus spike proteins were largely localized in endosomes (Fig. 2D). These data collectively suggest that SARS-CoV-2 enters into ECs through endocytosis, at least partially via caveolae, but is unable to export their RNAs from the endosome-like structure, and therefore cannot replicate in ECs.
Notably, both early passage and senescent HUVECs infected with SARS-CoV-2 appeared normal, and no significant cell death and morphological changes were detected at 72 hpi ( Supplementary Fig. 2). These data suggest that SARS-CoV-2 infection unlikely causes severe injury in ECs, and therefore, the severe endothelial injury detected in post-mortem lung analysis of COVID-19 may have been induced by excessive cytokines released by immune cells, but not by virus infection itself. We

SARS-CoV-2 infection affects various genes expressions in ECs.
We analyzed molecular events occurred in ECs after SARS-CoV-2 infection. Interestingly, genes involved in blood coagulation, leukocyte recruitment, and inflammation were upregulated in ECs after SARS-CoV-2 infection (Fig. 3A). Notably, these transcriptional changes were markedly enhanced in senescent HUVECs compared to those in early passage HUVECs (Fig. 3A). Because tissue factor (TF) is a potent inducer for blood coagulation, we further analyzed TF protein expression using immunocytochemistry. Significant TF-staining was observed in senescent HUVECs after SARS-CoV-2 infection, while TF-staining was very faint or undetectable in infected early passage cells ( Supplementary Fig. 4). These data suggest that SARS-CoV-2 infection in senescent ECs might cause enhanced blood coagulation through the high TF expression. Also, we assessed proliferation capacity in infected ECs, and found no effect of SARS-CoV-2 infection on proliferation capacity in both early passage and senescent HUVECs ( Supplementary Fig. 5).
We then performed comprehensive mRNA expression analyses by RNA-Seq. RNAs expression was analyzed in early passage and senescent HUVECs before and after SARS-CoV-2 or INFA infection at 1 MOI (at 72 hpi), and the alteration in gene expression was explored. Many genes expression was altered by virus infection, although the virus RNAs were mostly indetectable at this time point (Fig. 3B). Of note, the number of genes of which expression was significantly altered by virus infection was greater in senescent HUVECs than in early passage HUVECs in both cases of SARS-CoV-2 and INFA infection (Fig. 3B).
Gene ontology (GO) analysis for SARS-CoV-2 infection-related genes revealed 630 enriched GOs in early passage HUVECs, and 1047 enriched GOs in senescent HUVECs. When classified top 100 GOs by functions, different trend was observed in early passage and senescent HUVECs. In early passage HUVECs, more GOs related with cell cycle/proliferation and DNA process were detected, while GOs related with responses to stress and viruses were more prevalent in senescent HUVECs ( Supplementary Fig. 6). Similar number of GOs related with inflammation was detected in early passage and senescent HUVECs.
Venn diagram analysis showed that less than 50% of genes were commonly affected by each virus infection when compare between early passage and senescent HUVECs (Fig. 3C) (Fig. 3D). These data suggest that molecular events induced by virus infection in ECs varies depending on the types of viruses, and that cellular responses to the virus infection differ between early passage and senescent HUVECs. Pathway analysis for genes whose expression was significantly altered by twofold after the virus infection showed that various pathways were affected by the virus infection (Fig. 4). Pathways for inflammation and immune response were commonly affected, while the pathway for coagulation cascades showed up only in senescent HUVECs infected with SARS-CoV-2 (Fig. 4). These data strongly suggest a significant impact of SARS-CoV-2 infection in EC functions, and a possible contribution of the virus infection in ECs in the incidence of COVID-19-associated thrombosis, especially in elder patients.

Discussion
After the first report, COVID-19 has been immediately spread worldwide, and its pandemic is still uncontrolled. Striking feature of COVID-19 is the high mortality in elderly and the high incidence of systemic thromboembolisms. Because of the critical role of ECs in thrombosis, we explored the effects of SARS-CoV-2 infection in ECs using young early passage and replicative senescent cultured human ECs. Although post-mortem histological analysis of the lungs of COVID-19 patients showed severe endotheliitis, it was not conclusive whether SARS-CoV-2 indeed infects and injures ECs 8,26 . Furthermore, effects of SARS-CoV-2 infection on EC functions  Although the virus spike protein was not detected by immunostaining, virus RNA was detected in early passage HUVECs by quantitative PCR. Furthermore, many genes expression was altered in early passage HUVECs after exposure to SARS-CoV-2; therefore, we think SARS-CoV-2 indeed infects early passage HUVECs, though its infectivity seems to be quite low. These low infectivity and transient infection may cause the difficulties to detect  Our electron microscopic and immunocytochemistry analyses suggest that SARS-CoV-2 may enter into ECs through endocytosis. The best characterized endocytic routes are clathrin-and caveolae-mediated endocytosis 31 . Many viruses use these endocytic pathways to enter host cells [32][33][34] . It has been reported that SARS-CoV-2 enters the cells through clathrin-mediated endocytosis 35 , while contribution of caveolae-mediated endocytosis for SARS-CoV-2 infection remains unclear. Our electron microscopic data suggest that SARS-CoV-2 enters into ECs through caveolae-mediated endocytosis, and this pathway is remarkably enhanced in senescent ECs comparing to early passage cells. It has been reported that caveolin-1, the most important structural protein of caveolae, is upregulated in senescent cells 31 . Given that virus attachment was not different between early passage and senescent HUVECs, enhanced infectivity of SARS-CoV-2 in senescent ECs might be attributed to the senescenceassociated increase of caveolae-mediated endocytosis; yet, further analysis is certainly required to elucidate its underlying mechanisms.
Our data showed that SARS-CoV-2 remains and accumulates in endosomes even 48 hours after infection, and they disappear from the cells in 72 h after infection. These data suggest that SARS-CoV-2 is not able to export the genomic RNA into the cytosol, and therefore is unable to replicates in ECs. Accordingly, SARS-CoV-2 infection seems not to injure ECs as it dose in airway epithelial cells. Nevertheless, SARS-CoV-2 infection affected wide variety of genes expression in ECs. Cells sense the microbial infection largely through pattern recognition receptors such as Toll-like receptors (TLR), and some of TLRs are expressed at endosome to detect microorganisms entered through endocytosis 36 . Also, it has been reported that SARS-CoV-2 induces pro-inflammatory responses in macrophages via TLR-4 37 . Therefore, virus retention in endosome could lead to persistent activation of TLR-downstream signaling pathway such as NF-κB, resulting in the alteration of various genes expression. Further analyses are required to elucidate the signaling pathways through which SARS-CoV-2 infection alters gene expressions in ECs.
In this study, we used Wuhan strain of SARS-CoV-2 to analyze its infectivity in human cultured ECs. However, many variants that show different clinical features emerged, and the Wuhan strain is no longer a major strain for COVID-19; therefore, analysis for infectivity of SARS-CoV-2 variants in senescent ECs is needed, and  www.nature.com/scientificreports/ the analysis may provide mechanistic insights into variant-dependent unique clinical features. We used PR8 influenza A that is a mouse-adapted strain to explore the influenza infection in ECs in this study. It is notable that the findings using this strain cannot be generalized to other strains of influenza. Also, we used HUVECs to assess the virus infection in ECs in this study. Because the lungs are the major target tissue for SARS-CoV-2 infection, using ECs derived from lung microvessels, and compare the results with current study using HUVECs will provide additional important information.
In the current study, we revealed that cellular senescence enhances SARS-CoV-2 infection in ECs, and accentuates post infection molecular events including the modification of the pathway for coagulation. It has been reported that senolytics, which selectively kill senescent cells, reduced coronavirus-related mortality in old mice 38 , which support a crucial role of senescent ECs in the pathology of COVID-19. Analyses using young and aged mice infected with SARS-CoV-2 is a strong approach to observe infection-related microthrombi or barrier dysfunction in association with aging and/or cellular senescence, which should be done in the future. The sustained EC dysfunctions caused by SARS-CoV-2 infection may contribute to the microangiopathy in the lungs and to the prevalence of thromboembolisms in COVID-19, especially in elder patients in association with EC senescence.
Cell culture. Cells were regularly cultured in the CO 2 incubator for cells at 37 °C under 5% CO 2 levels.
Replicative senescent ECs were prepared as previously described 19 . Briefly, HUVECs were cultured in HuMedia-EG2 medium (Kurabo #KE-2150S), and regularly passaged at 1:4 ratio when reached sub-confluent. Cells were passaged until they did not show obvious proliferation with enlarged and flattened morphology (usually passage [18][19][20]. Cellular senescence was confirmed by enhanced senescence-associated β-GAL activity; reduced proliferation; increased expression of senescence-associated genes such as p16 and p21; accumulated DNA damage assessed by immunocytochemistry for γH2AX 19 . P3 HUVECs were regularly used as young early passage ECs. For virus infection, 1 × 10 5 cells were plated on 12-well plate, and incubated with SARS-CoV-2 (at MOI 1 or 50) or influenza A (at MOI 1) for 1 h with gentle rotation, followed by changing medium with fresh growth one. RNAs were extracted from the cells 6, 24, and 72 h after the infection. Attachment assay. SARS-CoV-2 attachment assay was performed as previously reported 39,40 . Briefly, early passage and replicative senescent HUVECs were detached from culture plates using versene solution, followed by washing with binding buffer (DMEM containing 2% FBS). Cells (2 × 10 5 ) were resuspended in 100 μl binding buffer, and then mixed with SARS-CoV-2 at 50 MOI. After incubation at 4 °C for 2 h, cells were washed twice with binding buffer to eliminate unbound viruses, followed by lysis in TRIzol (Invitrogen #15596026). Virus attachment was quantitatively analyzed using real-time PCR.
Quantitative PCR. RNAs were isolated from the cells using TRIzol, and then purified using NucleoSpin RNA clean-up kit (Macherey-Nagel #U0955). Subsequently, cDNA was synthesized using PrimeScript RT Master Mix (TaKaRa #RR036), followed by quantitative PCR using CFX384 (BioRad). Primers used were shown in Supplementary Table 1. The target gene mRNA and virus RNA expression levels were normalized to 18S expression levels. The relative gene expressions are presented in arbitrary units. RNAs were also subjected to RNA-Seq analyses. Library was prepared using TruSeq RNA Exome (Illumina #20020189), and paired-end RNA-Seq was performed using NovaSeq600 (Illumina). The data was analyzed using the iPathwayGuide (ADVAITA). The csCluster command of cummerbund was used to perform K-means clustering. Genes whose expression changes by more than 1.5-fold were included for analysis. Gene Ontology (GO) term analysis of gene clusters was performed using the goseq.
After measurements using DC protein assay kit (BioRad #500112), the same amount of proteins were run on SDS-PAGE gel, followed by transfer onto nitrocellulose membrane. The membrane was incubated with p21 (1:1000), or GAPDH (1:3000) antibody at 4 °C for overnight, following blocking with 10% skim milk. After washing with TBS-T, the membrane was incubated with HRP-labelled secondary antibody (1:2000), followed by detection using ChemiDoc Touch MP (BioRad).